Monday, February 05, 2007
Firefly Science - Luciferase and high throughput screening
Ah, fireflies - or 'Thunderbugs', as I idiomatically referred to them as a child. They are a staple of Summertime nostalgia, and a gateway insect for children to be drawn into the wonderful world of life science. A creature that can eat and create its own light source is something so alien and wonderful to us we are still exploring the science behind it. That said, Luciferase, the principle enzyme responsible for that soft yellow-green glow is a workhorse chemical tool in the high volume screening of compound libraries.
In screening, we often are looking for the activation of genes; that is to say whether the DNA is being transcribed into proteins, where those proteins then go out and perform the various functions of the cell ranging from the germane like cytoskeleton structure to the complex such as the release of a hormone or neurotransmitter. Every time a transcription event occurs, and we can exploit that mechanism for discovery research.
Originally, doing this required the use of radioactive tags - we could label a carbon in a designated molecule that could then be detected as a flash of light. Of course, radioactive work, despite its reliable and reproducible advantages, is not the prime choice of most research labs today.
A gene of interest can be appended downstream with the luciferase DNA and a gene promoter. Doing so allows us to infer that when our gene of interest is activated, our luciferase gene will be activated as well, and the cell will begin to produce luciferase in a ratiometric quantity, dependent on the strength of the promoter.
Now we can assess the amount of our gene of interest by lysing the cell, and adding the substrate luciferin. When this happens, the above reaction can take place, and a product of that reaction is light, which can be detected in the lab by a camera or photomultiplier tube (PMT). The more luciferase present, the more light the lysate will give off to be detected, and from that differential, we can begin to compare different treatments and draw conclusions from those results.
New sources of luciferase are being developed all the time. There's the firefly but there's a lot of different creatures out there with their own ways of giving off light. Mushrooms, sea-pansies and good ol' jellyfish that give us the standby Green Fluorescent Protein (GFP) With a series of enzymes that give off light at different wavelengths, one can create a very involved screen with multiple targets.